金黄色葡萄球菌凝固酶coa基因的克隆、表达及其生物活性分析  

Cloning,expression and biological activity analysis of coagulase gene coa of Staphylococcus aureus

作　　者：郭海勇[1] 赵雪[2] 朱小雨 赵研彤 林依丹 施爽 李铄 计银铎 宋勤叶[2] GUO Hai-yong;ZHAO Xue;ZHU Xiao-yu;ZHAO Yan-tong;LIN Yi-dan;SHI Shuang;LI Shuo;JI Yin-duo;SONG Qin-ye(School of Life Science,Jilin Normal University,Siping 136000,China;College of Veterinary Medicine,Agricultural University of Hebei,Baoding 071000,China;College of Veterinary Medicine,University of Minnesota,St Paul 55108,USA)

机构地区：[1]吉林师范大学生命科学学院,四平136000 [2]河北农业大学动物医学院,保定071000 [3]美国明尼苏达大学兽医学院,圣保罗55108

出　　处：《中国人兽共患病学报》2020年第9期696-702,共7页Chinese Journal of Zoonoses

基　　金：吉林省科技发展计划项目(No.20170101027JC)。

摘　　要：目的体外表达金黄色葡萄球菌(Staphylococcus aureus,S.aureus)凝固酶Coa,评价其生物学活性。方法根据GenBank中金黄色葡萄球菌凝固酶coa基因序列设计特异性引物,采用PCR方法从S.aureus USA300 CA-MRSA 923株基因组中扩增coa基因片段,将其克隆至pET-24b(+)表达载体,构建重组表达质粒pET-24b-coa;将经双酶切及测序鉴定正确的重组质粒转化感受态细胞E.coli BL21(DE3)中进行IPTG诱导表达;通过SDS-PAGE和Western blot对表达的重组Coa(rCoa)进行鉴定,测定rCoa对人血液和兔血浆的凝固活性;最后用纯化的重组蛋白rCoa免疫BALB/c小鼠,经间接ELISA和Western blot分析其免疫原性。结果构建的重组质粒pET-24b-coa在E.coli BL21(DE3)中以包涵体形式表达rCoa,分子量约74.8 ku,与预期蛋白大小相符;rCoa在体外具有凝固人全血和兔血浆活性,能刺激小鼠产生高效价的抗Coa特异性抗体。结论成功地获得了具有良好凝固酶活性和抗原性的金黄色葡萄球菌凝固酶重组蛋白rCoa,为进一步深入研究S.aureus凝固酶Coa的功能及以Coa为靶点的抗金黄色葡萄球菌抑制剂和疫苗的研制奠定基础。The object of this study is to express the coagulase(Coa)of Staphylococcus aureus(S.aureus)in vitro and evaluate its biological activity.According to the coa gene sequence of S.aureus in GenBank,a pair of primers was designed.DNA was extracted from S.aureus strain USA300 community-acquired methicillin resistant Staphylococcus aureus(CA-MRSA)923,and used as a template for PCR to amplify the coa gene.The coa gene was cloned into pET-24b(+)expression vector to construct the recombinant expression plasmid pET-24b-coa.After double digestion and sequencing analysis,the recombinant plasmids were transformed into competent cell E.coli BL21(DE3)and the coa gene was expressed by IPTG induction.The expression product was identified by SDS-PAGE and Western blot analysis.The coagulation activity of rCoa was measured in human whole blood and rabbit plasma in vitro,and the immunogenicity of rCoa was evaluated through detecting specific antibodies against rCoa in serum of BALB/c mouse immunized with purified rCoa by ELISA and Western blot.The results showed that rCoa,about 74.8 ku in line with expectations,was expressed in E.coli BL21(DE3)in the form of an inclusion body.The purified rCoa could coagulate human blood and rabbit plasma and stimulate mouse to produce high titer specific antibodies against Coa in serum.These results lay a foundation for further study on the function of S.aureus coagulase and the development of anti-infective agents and vaccine candidates targeting Coa.

关 键 词：金黄色葡萄球菌 凝固酶 生物活性 克隆表达 

分 类 号：R378.1[医药卫生—病原生物学] S852.61[医药卫生—基础医学]